Electron paramagnetic resonance (EPR) spectroscopy and electron spin resonance (ESR) spectroscopy are generally used to study molecular structure in chemistry, physics, biology, and medicine. EPR is also used to determine electron wave functions, lifetimes, and impurities in dielectrics used in solid state sciences. Prior EPR spectrometers comprise four main components: 1) a magnet to provide a steady DC magnetic field; 2) a high-Q microwave resonator in which a sample is placed; 3) a microwave bridge capable of producing an oscillating electromagnetic field which is coupled via a waveguide, coaxial cable, or the like to the resonator; and 4) a signal detector with field modulation, signal amplification and display systems.
In EPR, a steady magnetic field is applied to the chemical sample in the microwave resonator. The steady magnetic field causes the electrons in the sample to precess at a frequency defined by the sample composition. The precession frequency is called the paramagnetic resonant frequency and is proportional to the intensity of the applied steady magnetic field. The precession is altered by application of high frequency energy when the frequency of the energy is near the paramagnetic resonance frequency. If the sample contains unpaired electrons, the precession change can be detected. A comparison of change in electron precession as a function of magnetic field or frequency provides valuable information relating to the chemical characteristics of the sample.
The process of detection of a resonance signal involves the detection of the absorption of electromagnetic energy from a sample containing unpaired electron or nuclear spins. These spins are prepared in a magnetic field that selectively aligns the spins parallel or anti parallel relative to the preparative magnetic field; although they also respond to environmental magnetic fields. The spins couple to the magnetic fields via the magnetic moment ineluctably produced by their spins. The spins preferentially align either with or against the magnetic field producing a net magnetization of the sample, a macroscopic quantity that can absorb electromagnetic energy by which the magnetization and the spins of which it consists can be detected. This absorption takes place at a specific frequency proportional to the sum of the magnetic field produced by the preparative magnet and the environmental magnetic fields. In addition to the variation of the total magnetic field from the environment, there is a variation or spectrum of frequencies at which the absorption occurs due to lifetime broadening, an effect described by quantum mechanics. The detection of this absorption as a function of frequency can involve apparatus involving either a highly tuned system amplifying a magnetic absorption signal at a specific microwave- or radio-frequency while the preparative magnetic field is swept. The result is a spectrum of the conditions of the magnetization, which can be imaged.
Another highly sensitive method of detection is to subject the magnetization to a very short high power pulse of microwave- or radio-frequency electromagnetic energy. The short duration of the pulse means that it contains a wide band of frequencies. Ideally, this band of frequencies spans the spectrum of the magnetization. The duration of the pulse multiplied by the square root of the power of the pulse determines the angle through which the magnetization, initially oriented parallel or antiparallel to the preparative magnetic field, rotates relative to its initial direction. Having been so rotated, the magnetization precesses or rotates perpendicular to the preparative magnetic field. This precessing magnetization creates a time varying electromagnetic field of its own. This can be detected by a resonator.
A typical EPR spectrometer uses a reflection type measurement on the electrical resonator that contains the sample. In a reflection type spectrometer a single resonator is used. The sample is placed in the resonator and microwave energy is injected via a waveguide, coaxial cable, or the like into the resonator while the sample and resonator are positioned in the steady magnetic field. A microwave device called a circulator is often used to separate the desired EPR signal from the microwave source power. A disadvantage of the reflection mode of operation is that any portion of the microwave source power that is reflected from the resonator will interfere with the EPR signal generated in the resonator. In one type of measurement, also called “continuous wave” because the input microwave energy is applied as a continuous AC signal, the resonator is tuned to provide minimal reflection of the input energy when the input frequency is different from the paramagnetic resonance frequency.
The amplification provided by the resonant response of a resonator is proportional to its quality factor or Q. The Q is defined of the central tune frequency of the resonator divided by the half width at half maximum of the profile of the frequencies to which it can respond. The larger the Q and the smaller its frequency band pass, the higher the amplification and the more signal that can be detected from the precessing magnetization.
There is a problem in pulse measurements with high Q resonators. The time over which the initial, exciting pulse energy remains in the resonator is also proportional to the Q. This exciting pulse energy blinds the detection apparatus to the small magnetization signal.
Traditionally, magnetic resonance pulse experiments or magnetic resonance pulse images simply wait until the exciting pulse has dissipated in the detection apparatus before detection is begun. This is referred to as the “dead time”, because it is necessary to desensitize the detection apparatus or deaden it while waiting for the excitation pulse to dissipate. However, if this dead time is too long a time interval, the magnetization itself may disappear via physical mechanisms that dissipate the coherence and the energy in the magnetization itself. There are many circumstances and substances that make detection and imaging impossible because the dead time exceeds the lifetimes of the magnetization signal.
An approach to this problem is to change the Q of the resonator very rapidly. Immediately after the exciting pulse signal, the Q is electronically spoiled or switched to a much lower value using diode switches that rapidly lowers a resistance in parallel to the resonator circuit. This shunts the excitation pulse power from the resonator to a load and much more rapidly reduces the pulse power in the resonator and shortens the time over which the detection system needs to be deadened. This in turn allows more rapid onset of detection and allows more transient signals to be detected and imaged.
Heretofore, Q spoiling has been an active process. By this we mean that external voltage signals are used to bias the diodes into states wherein they conduct current into the load resistors (lowering the Q) or block such conduction into the load resistors (increasing the Q). The external switching has its own onset time and can affect the detection system as well.
One disadvantage of reflection type spectrometers for continuous wave (CW) measurements is that the EPR signal is minute compared to the magnitude of the injected microwave energy. The signal detector must detect the EPR signal while separating out the injected microwave energy. It has proved difficult to completely separate the EPR signal from the input power.
Another difficulty arises in that any parasitic reflection of the microwave source caused by improper coupling of the input power to the resonator creates significant noise in the EPR signal. In addition, the source input waveguide and the detector waveguide must be critically coupled to the resonator to prevent a large reflection of the input power that would add to the EPR signal and saturate the detector electronics.
Phase noise or noise frequency modulation of the microwave source is converted to noise amplitude modulation in the reflected signal by the resonator, creating further noise in the EPR signal. Phase noise cannot be eliminated from microwave sources. It can be reduced but this results in higher costs. Since the phase noise intensity is proportional to the source intensity, it becomes more serious at higher powers. Hence, current EPR tools must be operated at low power which in turn requires larger samples. A phase or dispersion component of the reflected EPR signal is difficult or impossible to study in reflection-type spectrometers because of this phase noise.
EPR tools can also be used for pulse-type measurements such as electron spin echo (ESE). In pulse type measurements, the input energy is provided by a high power pulse rather than a continuous wave microwave source. The pulse causes a near instantaneous change in the precession and a gradual decay as the sample returns to the baseline state created by the DC magnetic field. In this type of measurement the difficulty in separating input power from the EPR signal requires a delay after the application of the input pulse before a measurement can be made. Because the energy stored in the resonator by the input pulse must “ring-down” or dissipate before a measurement can be taken, much of the ESE signal can be lost in a reflection-type spectrometer.
A common type of resonator used in EPR spectrometry is the cavity resonator. Cavity resonators were used in early spectrometers due to their easily modeled performance, availability, and high Q. Cavity resonators are called distributed element circuits because the microwave, magnetic, and electric field are continuously distributed and mix throughout the cavity. Characteristic dimensions of cavity resonators are of the same order of magnitude as the wavelength of the electromagnetic fields used. More recently, lumped element resonators have been suggested because their dimensions can be much smaller than the wavelengths of interest.
These conventional cavity resonators are typically ill suited for the study of lossy dielectric samples, which includes most biologicals and solutions of free radicals. The sample volumes of lossy living tissue utilized by the conventional cavity resonators are measured by the tens of microliters because high frequency microwaves suitable for resonators are absorbed in larger volumes. However, biological samples are often limited in supply which presents a particularly troublesome problem since employing a conventional cavity resonator to study transient processes usually requires large volumes of relatively concentrated material. Another problem arises from the failure of the conventional cavity resonator to effectively isolate the region of microwave electric field (E1) from the region of microwave magnetic fields (H1), the latter of which induces the desired EPR transitions. The inability to separate the Eland H1 components is an important characteristic since the electric field may often interact with a sample to cause resonant frequency changes and Q losses (Q is the quality factor, either calculated as being 2 pi times microwave energy stored by the device/energy dissipated per cycle of microwaves or calculated as the resonant frequency (υ0) of the device/the difference in frequency (Δυ) obtained at the 3 dB half power absorbing points on the mode pattern of the device). This undesirable interaction between the sample and the E1 component is especially pronounced with lossy dielectric samples.
The Alderman-Grant resonator (AGR) is a popularly used saddle coil resonator used in water proton nuclear magnetic resonance (NMR) based MRI for localized imaging, although it can be used for whole body imaging. The resonator produces a radiofrequency or microwave magnetic field perpendicular to the axis of the cylindrical sample container. One of the attractive characteristics of the Alderman-Grant design is the excellent containment of electrical fields within the structures of the resonator, shielded from the sample in the cylindrical sample container while producing a time varying (oscillating) magnetic field to excite nuclear or electron spins in the sample. This time varying magnetic field generates magnetization in the sample. The corresponding time varying electric fields generate energy loss and shift in resonator tune that interferes with measurements. By containing the electric fields within the resonator structures, the Alderman-Grant type resonator is a very stable magnetization generator and detector.
A design more recently used for continuous and stopped flow EPR is based on a loop gap resonator (LGR) as described in Froncisz and Hyde (1982). The standard design for an LGR utilizes a machined MACOR® ceramic block having two holes extending through the block, these holes are connected by a thin slit extending through said block, the interior of the holes and slit are plated with silver. Unlike the conventional cavity resonators the LGR utilizes a much smaller sample volume; however, due to the complex configuration of the LGR and its small components the LGR based EPR probe is typically susceptible to a significant loss of sensitivity with use. In addition, due to the configuration of the loop and gap areas of the LGR low Q is experienced due to electric field (E1) interaction with lossy dielectric samples. In addition, due to the design of the LGR, flow and stopped-flow induced noise is a limiting factor when utilizing stopped flow technology since repetitive starting and stopping of the sample flow in the capillary is required. This forced movement within the capillary tube creates vibrations which effectively limit the sensitivity of the LGR. In addition, the structure of the LGR makes it difficult to assemble and disassemble the device. In the event any particular part becomes contaminated or worn, the ability to replace or repair any individual component takes considerable effort and often requires returning the part to the manufacturer. In addition, the use of delicate and complex machined parts results not only in less durable parts but in expensive replacement parts. Furthermore, variable capacitance coupling used in connection with the LGR probe often causes large resonance frequency changes when the coupling is changed. The resulting simultaneous coupling and frequency changes greatly complicate attaining critical coupling
One means that has been tried in order to reduce the problems associated with reflection type resonators is a bimodal resonator. The development of a practical bimodal resonator for EPR has been sought for over 20 years. A bimodal cavity resonator was commercially available from Varian Associates, Inc., but suffered from complex and difficult tuning requirements related to the cavity resonator design. Most recently, a bimodal loop gap resonator was investigated for EPR spectroscopy. In 1992 A. I. Tapin, James S. Hyde, and W. Froncisz published a paper entitled Bimodal Loop-Gap Resonator in the Journal of Magnetic Resonance 100, 484-490 that proposed a loop-gap resonator in which the two orthogonal EPR modes did not overlap in some regions of space but overlapped and were orthogonal in the sample-containing region. Unfortunately, a commercially viable implementation has not been produced. A need exists for a resonator structure for EPR spectroscopy that effectively isolates the input power from the detector yet is easy to tune and inexpensive to build.
The limitations of prior resonator structures are a primary impediment to the application of EPR spectroscopy to biology and biomedical research. Biological and biomedical applications of EPR spectroscopy are limited by low signal-to-noise resulting from the small number of spins in the sample and instrumental sources of noise, e.g., microwave source noise, magnetic field modulation, detector noise, and, in time-domain EPR, by the dead-time of the system after the microwave pulse. Because EPR is able to detect and analyze “free radicals” and metalloenzymes either naturally occurring or used as labels or probes, overcoming these impediments to EPR spectroscopy for biological samples has important commercial and scientific significance.